Methods of treatment using a pentapeptide derived from the C-Terminus of Glucagon-Like Peptide 1 (GLP-1)

ABSTRACT

Methods of treating obesity, metabolic syndrome, hepatic and non-hepatic steatosis, and diabetes using a pentapeptide, LVKGRamide, derived from the C-terminus of Glucagon-Like Peptide 1 (GLP-1).

CLAIM OF PRIORITY

This application is a continuation of U.S. patent application Ser. No.14/128,801, with a 371 filing date of Mar. 21, 2014, which is a U.S.National Phase Application under 35 U.S.C. § 371 of International PatentApplication No. PCT/US2012/045537, filed on Jul. 5, 2012, which claimsthe benefit of U.S. Provisional Patent Application Ser. No. 61/504,866,filed Jul. 6, 2011, and Ser. No. 61/546,698, filed on Oct. 13, 2011, theentire contents of the foregoing are hereby incorporated by reference.

TECHNICAL FIELD

This invention relates to methods of treating obesity, metabolicsyndrome, hepatic and non-hepatic steatosis, and diabetes using apentapeptide, LVKGRamide, derived from the C-terminus of Glucagon-LikePeptide 1 (GLP-1).

BACKGROUND

The prevalence of obesity-related metabolic syndrome consisting ofdiabetes, hypertension, hypertriglyceridemia, hepatic steatosis, andaccelerated atherosclerosis is increasing worldwide. No satisfactorytreatments are available for obesity and the metabolic syndrome.

SUMMARY

At least in part, the present invention is based on the discovery that apentapeptide, GLP-1(32-36)amide (LVKGRamide (SEQ ID NO:1)) derived fromthe C-terminus of the glucoincretin hormone GLP-1 curtails thedevelopment of obesity, insulin resistance, diabetes,hypertriglyceridemia, and hepatic steatosis, and increases energyexpenditure.

Thus, in a first aspect, the invention provides isolated peptidesconsisting essentially of a sequence Leu-Val-(Lys/Arg)-Gly-Arg-Xaa (SEQID NO:3), wherein Xaa can be Gly, Gly-Arg, Gly-Arg-Gly, or absent.

In some embodiments, the peptide is amidated. The lysine (K) andarginine (R) in LVKGRamide are in a configuration potentially acceptablefor acetylation. Amidation occurs at the C-terminal amino acid of somepeptides. In some embodiments, when the C terminus is an Arg, thearginine is amidated. In some embodiments, e.g., the correspondinghexapeptide, LVKGRG, the G, glycine, is not amidated. In someembodiments, one or more amino acids are modified by attachment of afatty acid, e.g., palmitate or oleate.

In a further aspect, the invention provides fusion peptides comprising afirst portion consisting essentially of a sequence:Leu-Val-(Lys/Arg)-Gly-Arg-Xaa (SEQ ID NO:3), wherein Xaa can be Gly,Gly-Arg, Gly-Arg-Gly, or absent, fused to a cell-penetrating peptide. Insome embodiments, the cell-penetrating peptide is fused on theC-terminus of the peptide. In some embodiments, the cell-penetratingpeptide is fused on the N-terminus of the peptide. In some embodiments,the cell-penetrating peptide is selected from the group consisting ofHIV-derived TAT peptide, penetratins, transportans, SS peptides, and hCTderived cell-penetrating peptides.

In yet another aspect, the invention provides isolated nucleic acidsencoding the peptides or fusion peptides described herein, and hostcells including and/or expressing the isolated nucleic acids.

In an additional aspect, the invention provides therapeutic compositionsincluding the peptides or described herein in a physiologicallyacceptable carrier. In some embodiments, the compositions furtherinclude at least one cell-penetrating agent, e.g., a cationic liposome.

Also provided herein is the use of the peptides or fusion peptidesdescribed herein in the treatment of obesity or an obesity-relateddisorder. In some embodiments, the obesity-related disorder is diabetesor the metabolic syndrome, hepatic steatosis, non-hepatic steatosis orhypertriglyceridemia.

In yet another aspect, the invention features methods for treatingobesity or an obesity-related disorder in a subject. The methods includeadministering a therapeutically effective amount of a peptide or fusionpeptide as described herein. In some embodiments, the obesity-relateddisorder is diabetes or the metabolic syndrome, hepatic steatosis,non-hepatic steatosis or hypertriglyceridemia.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIGS. 1A-B show that GLP-1-derived pentapeptide, GLP-1(32-36)amide(LVKGRamide) (SEQ ID NO:1) curtailment of weight gain in diet-inducedobese mice. The data encompass twelve weeks, from week 2 to week 14 ofcontinuous infusions of control vehicle and GLP-1(32-36)amide.GLP-1(32-36)amide was administered to mice via mini-osmopumps (Alzet#1004) at a rate of 40 nanomoles/Kg body weight/24 hours. Male C57bl/6jmice were placed on a high fat diet (Research Diets, #D12492, 60 kcal %fat) at six weeks of age and continued for 17 weeks before startingosmopump infusions at age 23 weeks. 1A. Line graph showingtime-dependent changes in body weight (BW) over fourteen weeks ofinfusions of control vehicle and pentapeptide GLP-1(32-36)amide. (*)p<0.05 versus vehicle control; (**) p<0.04 versus vehicle control; (***)p<0.034; (#) p<0.01; (##) p<0.004; (###) p<0.0006, 0.0007; (####)p<0.0001. 1B. Bar graph showing average weekly weight changes over tenweeks of infusions (weeks 2 to 14) shown in FIG. 1A. N=six mice pergroup. Values are means+/−SEMs. *p<0.013 peptide vs. vehicle. At weektwo of infusions of vehicle and pentapeptide the mice weighed 37.2 and38.2 gms (averages of six mice), respectively. N=six mice per group.

FIGS. 2A-D are bar graphs showing the results of determinations of lean,fat, and total body mass by dual X-ray absorptiometry (DXA) of micereceiving continuous infusions of the pentapeptide GLP-13(2-36)amide orcontrol vehicle alone for 16 weeks. At the time of the study the micewere 39 weeks of age and on the very high fat diet for 33 weeks. 2A. Fatversus lean mass. The fat mass is 40% less in the pentapeptide infusedcompared to the control vehicle infused mice. *P<0.0008. There is nosignificant difference in the lean mass between the two groups ofinfused mice. 2B. Total body mass (lean+fat) differences in peptideinfused versus vehicle control infused obese mice. *P<0.002. 2C. Fatmass expressed as the fraction of fat mass in peptide infused miceversus vehicle control infused obese mice. *P<0.0008. 2D. Fat massexpressed as the percentage of body weight, peptide-infused versusvehicle control-infused obese mice. *P<0.001. N=6 mice per group. Valuesare means+/−SEMs.

FIG. 3 is a bar graph showing feed efficiency at the end of 12 weekinfusion. GLP-1(32-36)amide infusions in diet-induced obese mice lowersthe Feed Efficiency index. The data are the means+/−SEMs (*p<0.06pentapeptide vs. vehicle) of the values calculated from the ten weekperiod of infusions of control vehicle and GLP-1(32-36)amide shown inFIG. 4. The Feed efficiency (FE) is a calculation of the body weight(gms) divided by the energy intake kcal) in the food consumed. The lowerFE in the mice receiving the pentapeptide GLP-1(32-36)amide compared tothat of control vehicle indicates that less energy is going into weightand instead is expended. Basal energy expenditure in mice treated withpentapeptide is increased compared to mice treated with control vehicle.N=6 mice per group.

FIGS. 4A-C show the results of metabolic measurements of body weights,energy expenditure (VO2), and physical activities of mice during threedays of monitoring in TSE-Systems metabolic cages. Mice were 37 weeksold and fed the very high fat diet for 31 weeks at the time of thestudy. 4A. Line graph showing body weights during three-day monitoring.Vehicle N=2; GLP-1s N=3. # p<0.01; ## p<0.006; ## p<0.008; ## p<0.007.4B. Bar graph and line graph showing total body oxygen consumption (VO2)measured during both light and dark cycles for the three days ofmonitoring. Upper panel; Total VO2. Lower panel: Daily time-course ofVO2 during light and dark cycles averaged over the three days. VehicleN=2; GLP-1s N=3. 4C. Bar graph and line graph showing physical activityof the mice during the three days of the metabolic studies. Upper panel:Total activities averaged for light and dark cycles. Lower panel:Time-dependent measurements of activities. Activities were measured forhourly intervals during the light cycle of six hours (13:00 to 18:00)and for two hour intervals during the dark cycle of 12 hours (18:00 to6:00). The differences in body weights and total VO2 were highlysignificant for GLP-1(32-36)amide versus control vehicle treated mice,whereas there were no significant differences in total activity betweenthe two groups of mice (light cycle, p<0.4; dark cycle, N.S.). VehicleN=2; GLP-1s N=3. The findings support a model of increased basal energyexpenditure in mice infused with GLP-1(32-36)amide versus mice infusedwith control vehicle, independent of endogenous energy expenditure dueto physical activity. N=3 mice for pentapeptide infusion and N=2 micefor control vehicle infusion.

FIGS. 4D-G show the results of metabolic measurements of energyexpenditure (VO2) of monitoring in TSE-Systems metabolic cages. Micewere 33-34 weeks old and fed the very high fat diet for 23-24 weeks atthe time of the study. 4D and 4E. Line graph and bar graph showing totalbody oxygen consumption (VO2) measured during the light phase during twodays. 4F and 4G. Line graph and bar graph showing lean body oxygenconsumption (VO2) measured during the light phase during two days.

FIGS. 5A-B are a pair of bar graphs showing that GLP-1(32-36)amideattenuates the development of fasting hyperglycemia and hyperinsulinemiain high fat-fed mice. Mice fed the VHFD develop hyperglycemia,hyperinsulinemia, diabetes, and insulin resistance during weeks 15 to 29of diet-induced obesity (21-35 weeks of age, respectively). A 12-weekinfusion of either control vehicle or pentapeptide started at 17 weekson high-fat diet (23 weeks of age). FIG. 5A: Fasting plasma glucoselevels *p<0.0004 vehicle vs. GLP-1(32-36)a. FIG. 5B: Fasting insulinlevels *p<0.042 vehicle vs. GLP-1(32-36)a. The studies are in mice fed aVHFD for 15 weeks, two weeks before beginning the continuous infusionsof either control vehicle or pentapeptide. Continuous infusions ofvehicle or pentapeptide GLP-1(32-36)amide were give during weeks 17 to29 of diet (23 to 35 weeks of age). Weeks denoted on the ordinate scalesrefers to weeks of age. N=6 mice per group. Values are means+/−SEMs.

FIGS. 5C-D are a pair of line graphs Plasma glucose levels (FIG. 5C) and(FIG. 5D) Plasma insulin levels after an intraperitoneal glucoseadministration (2 mg/Kg BW ip) in overnight fasted mice fed in VHFD for24-weeks and 16-weeks of continuous infusions of vehicle orGLP-1(32-36)amide. ***p<4E-03 vehicle vs. GLP-1(32-36)amide, *p<0.01vehicle vs. GLP-1(32-36)amide, **p<0.004 vehicle vs. GLP-1(32-36)amide,*****p<1.7E-05 vehicle vs. GLP-1(32-36)amide, ****p<6.7 E-04 vehicle vs.GLP-1(32-36)amide.

FIGS. 6A-B are bar graphs showing that infusions of GLP-1(32-36)amide inmice prevent the accumulation of triglyceride in the livers of mice feda very high-diet (VHFD). Triglyceride contents of samples of livers ofmice fed a high-fat diet (VHFD) for 32 weeks (age 38 weeks). 6A.Triglyceride levels of pentapeptide-infused obese mice as a percent ofthe vehicle-infused control mice set at 100%. *p<0.005. 6B. Triglyceridecontent levels of 60.5+/−12.1 mg/mg protein. Infusion of thepentapeptide lowered the triglyceride content by 65% to 21.1+/−5.6 mg/mgprotein, indistinguishable from the values in the livers of lean micefed low-fat chow (21.6+/−7 mg/mg protein). Data from diet-induced obesemice infused with the nonapeptide, GLP-1(28-36)amide for 16 weeks arealso shown. Values are represented as % of vehicle control infused micefed a high-fat diet (VHFD). *p<0.005, peptide vs. vehicle. N=6 mice pergroup. Values are means+/−SEMs.

FIG. 6C is a table showing continuous infusion of GLP-1(32-36)amide for16-weeks improves plasma circulating levels of glycerol and triglyceridein mice fed on a 60% fat diet (VHFD) for 24-weeks. *p<0.02, peptide vs.vehicle; **p<0.008 peptide vs. vehicle.

FIG. 7A is a diagram of the amino acid sequence of GLP-1(7-36)amide[HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRamide (SEQ ID NO:7)], the insulinotropicform of GLP-1 that augments glucose-sensitive insulin secretion, showingthe sites cleaved by the two endopeptidases, Dpp4 and NEP. Dpp4 is adiaminopeptidyl peptidase that removes the first two amino acids ofGLP-1, resulting in the formation of GLP-1(9-36)amide devoid ofinsulin-releasing activities and currently believed to be an inactivemetabolite of GLP-1. This belief has become controversial sinceGLP-1(9-36)amide has been shown to have insulin-like actions on insulinresponsive peripheral (extrapancreatic) tissues. [9-15] NEP, anabbreviation for NEP 24.11, is an endopeptidase, known as neprilysin,CALLA, CD10, that selectively cleaves on the amino-proximal side ofhydrophobic amino acids such as the E-F site and the W-L site in GLP-1generating the nonapeptide, GLP-1(28-36)amide [FIAWLVKGRamide (SEQ IDNO:2)] and the pentapeptide, GLP-1(32-36)amide [LVKGRamide (SEQ IDNO:1)], respectively. The overwhelming current belief is that NEPcompletely degrades GLP-1 and is involved in its disposal [5, 6]. Thisbelief is challenged by recent publications [17, 18] and the findingsreported herein manuscript. The present results support the hypothesisthat NEP is not a degrading enzyme for GLP-1. Rather the evidencesupports the concept that NEP purposefully cleaves GLP-1, in particularthe pro-peptide GLP-1(9-36)amide, to generate the two bioactivepeptides, the pentapeptide and the nonapeptide, that have insulin-likeand antioxidant-like actions on insulin resistant, stressed cells.

FIG. 7B is a line graph showing the results of experiments involvingintravenous push injection of GLP-1(9-36)amide, 32-36 amide, and 38-36amide into mice, collecting blood plasma at 2, 5, and 10 min, andanalyzing the peptide profiles by liquid chromatography-massspectrometry (LC-MS). Relative amounts of peptides at times indicatedare shown. Y-axis, arbitrary units

FIG. 7C shows data from LC-MS analyses of mouse plasma samples followingi.v. injection of GLP-1(9-36)amide as described in FIG. 7B. The LC-MSprofiles of the 2 min plasma sample shows the presence of the twopeptides, 32-36 amide and 28-36 amide that could only have beengenerated by endopeptididic cleavages from the GLP-1(9-36)amidepro-peptide that was injected into the mice.

FIG. 8 is a bar graph showing suppression of glucose production by thepentapeptide GLP-1(32-36)amide (LVKGRamide (SEQ ID NO:1)) ininsulin-resistant hepatocytes. Mice (male, C57bl/6) from 12-19 weeks ofage were fasted overnight (16 hrs) and primary hepatocytes were isolatedusing a collagen and perfusion gradient purification [15]. Cells werefirst seeded using a Dulbecco's modified Eagle's medium (DMEM)supplemented with 10% FBS, 1 g/L glucose, 2 mmol/1 sodium pyruvate, 1micromol/L dexamethasone, and 0.1 micromol/L insulin, and latermaintained in DMEM with 0.2% BSA, 1 g/L glucose, 0.1 micromol/Ldexamethasone, and lnmol/L insulin. After an overnight incubationprimary hepatocytes (2×10⁵ cells per well in twelve-well plates) werepre-treated with GLP-1(32-36)amide for 1 hour followed by stimulationwith cAMP (10 microM)/dexamethasone (50 mM)/sodium lactate (2 mM) inglucose-free DMEM without phenol red containing the same concentrationsof pentapeptide. After 2.5 hrs the culture media were collected formeasuring glucose concentration with a colorimetric glucose assay kit(Sigma). The readings were then normalized to total protein contentdetermined from whole-cell lysates. The data shown are the means+/−SEMsof three separate hepatocyte isolations.

FIG. 9A is a bar graph showing suppression of the production of reactiveoxygen species (ROS) by the pentapeptide GLP-1(32-36)amide ininsulin-resistant isolated mouse hepatocytes. Hepatocytes were renderedinsulin-resistant by their pre-incubation in 30 mM glucose. Primaryhepatocytes from C57BL/6J mice were seeded at 2×10⁵ cell per well intwelve-well plates and treated for 24 h with 30 mM glucose in theabsence of presence of GLP-1(32-36)amide (10 pM, 100 pM and 100 nM). Tomeasure ROS levels cells were washed twice in HBSS (Hanks' Balance SaltSolution) and incubated with 10 uM 5-(and6)-carboxy-2′,7′-dichlorohydro-fluorescein diacetate (CM-H₂DCFDA)(Molecular Probes) for 45 minutes. The media were removed and cells werelysed. ROS was measured in the cell lysates using a spectrofluorometer(485 nm/535 nm). Data were normalized to values obtained from untreatedcontrols. The data shown are the means of two independent hepatocyteisolations+/−SEMs.

FIG. 9B is a bar graph showing inhibition of the production of ROS inH4IIe hepatoma cells. BSA n=14. palmitate n=13. 32-36/palmitate n=12.*p<0.005 palmitate vs BSA, # p<0.03 palmitate vs GLP-1(32-36)a/palmitate

FIG. 10 is a schematic illustration of a hypothetical model of theproposed mechanism of GLP-1 action on hepatocytes. The mechanismproposed does not involve the known GLP-1 receptor. The majorcirculating form of GLP-1 are theorized to be GLP-1(32-36)amide andGLP-1(28-36)amide [8] formed by the cleavage of GLP-1(9-36)amide in thecirculation by an endopeptidase such as NEP 24.11 or a relatedendopeptidase. The GLP-1(32-36)amide is transported into hepatocytes byan as yet unidentified mechanism such as by a novel receptor ortransporter. After entry into the hepatocyte GLP-1(32-36)amide targetsto mitochondria, gains access to the mitochondrial matrix, and modulatesoxidative phosphorylation, gluconeogenesis, energy production, andcellular redox potential. It is proposed that the inhibition ofoxidative stress by GLP-1(32-36)amide might have cytoprotective(anti-apoptosis) effects on hepatocytes.

DETAILED DESCRIPTION

Obesity-related metabolic syndrome is manifested as diabetes,hypertension, hyper-lipidemia, hepatic steatosis, and acceleratedatherosclerosis. The pathophysiology underlying the metabolic syndromeis the development of insulin resistance and resulting increasedoxidative stress [1,2]. Currently, effective treatments for obesity andmetabolic syndrome are not available. As described herein, aGLP-1(32-36)amide pentapeptide (LVKGRamide) (SEQ ID NO:1) derived fromthe C-terminus of the glucoincretin hormone glucagon-like peptide-1(GLP-1) curtails weight gain and the development of the metabolicsyndrome in an animal model of diet-induced obesity.

GLP-1 is a glucoincretin hormone that augments glucose-dependent insulinsecretion. GLP-1 receptor agonists are in use for the treatment of type2 diabetes based on their stimulation of insulin secretion and alowering of plasma glucose and HgbA1C levels [3,4]. It is generallybelieved that the full-length receptor agonist forms of GLP-1, such asGLP-1(7-36)amide, are rapidly inactivated in the circulation viacleavages by the endopeptidases diaminopeptidyl peptidase-4 and byneprilysin, a neutral endopepdidase (NEP 24.11) known as neprilysin[5,6]. The removal of the first two amino acids of GLP-1(7-36)amidegives rise to GLP-1(9-36) amide devoid of insulin-releasing activitiesand NEP 24.11 cleaves GLP-1 into several small peptides [7]. However, itwas postulated earlier that cleavages of GLP-1 by the endopeptidasesDpp4 and neprilysin do not degrade the peptide but rather generate newC-terminal peptides with insulin-like actions on insulin-responsivetarget tissues [8]. Recent evidence indicates that GLP-1(9-36)amide, theproduct of cleavage of GLP-1 by Dpp4, exerts insulin-like andanti-oxidant cytoprotective actions on heart, vasculature, and liver[9-16]. Infusions of GLP-1(9-36)amide in obese, insulin-resistant humansubjects suppresses hepatic glucose production without effect on plasmainsulin levels [14]. Continuous infusion of GLP-1(9-36)amide for eightweeks in diet-induced mice curtails weight gain, increases energyexpenditure, and inhibits the development of insulin resistance,diabetes and hepatic steatosis [16]. Furthermore, Infusions of thenonapeptide, GLP-1(28-36)amide, a product of the cleavage of GLP-1 byneprilysin [7], in diet-induced mice inhibits weight gain, thedevelopment of diabetes, hepatic steatosis, and increases energyexpenditure [17]. The nonapeptide was found to enter isolatedinsulin-resistant mouse hepatocytes in vitro, target to mitochondria,and to suppress glucose production, reactive oxygen species, and toincrease cellular ATP levels [18]. Notably, the actions of theGLP-1-derived C-terminal peptides, GLP-1(9-36)amide andGLP-1(28-36)amide appear to occur selectively in obese,insulin-resistant conditions and not in lean, insulin-sensitive humansubjects [14] or mice [17] and occur by mechanisms independent of theGLP-1 receptor. Therefore, much new evidence indicates thatGLP-1-derived peptides exert extrapancreatic actions oninsulin-resistant tissues independent of the GLP-1 receptor [8,19].

GLP-1 C-Terminal Peptides, Fusion Peptides, Peptidomimetics, andModifications

The GLP-1 C-terminal peptides described herein include the sequenceLVKGRamide (SEQ ID NO:1), or a variant thereof. Variants includepeptides in which the sequence is C-terminally extended, e.g., LVKGRG(SEQ ID NO:4), or LVRGRG (SEQ ID NO:5), or in which one or more aminoacids are conservatively substituted, for example LVRGRamide (SEQ ID NO:6), in which Lysine 34 (the numbering refers to the full-length GLP-1)is changed to Arginine. In some embodiments the peptides also includethe sequence FIAW on the N-terminus. Methods for making these peptidesare known in the art, e.g., using chemical synthesis or expression in ahost cell.

Fusion Peptides

In some embodiments, the peptides also include a cell-penetrating moietythat facilitates delivery of the peptides to the intracellular space,e.g., HIV-derived TAT peptide, penetratins, transportans, SS peptides(alternating aromatic residues and basic amino acids (aromatic-cationicpeptides)), SA, SM, or SNL peptides, or hCT derived cell-penetratingpeptides, see, e.g., Caron et al., (2001) Mol Ther. 3(3):310-8; Langel,Cell-Penetrating Peptides: Processes and Applications (CRC Press, BocaRaton Fla. 2002); El-Andaloussi et al., (2005) Curr Pharm Des.11(28):3597-611; Lindgren et al., Trends Pharmacol Sci. 21(3):99-103(2000); Zhao et al., J Biol Chem 279:34682-34690 (2004); Szeto, AAPSJournal 2006; 8 (2) Article 32; Deshayes et al., (2005) Cell Mol LifeSci. 62(16):1839-49; Horn et al., J Med. Chem., 46:1799 (2003); Bonny etal., Diabetes, 50:77-82 (2001), and U.S. Pat. Nos. 6,841,535 and7,576,058 and references cited therein. In some embodiments thecell-penetrating moiety is linked to the peptide, e.g., as a singlefusion protein; thus, the invention includes fusion proteins comprisinga GLP-1 C-terminal peptide as described herein and a cell-penetratingpeptide, e.g., TAT, penetratins, transportans, or hCT derivedcell-penetrating peptides. In some embodiments, the cell-penetratingpeptide is attached to the N-terminus of the GLP-1 C-terminal peptide;in some embodiments, the cell-penetrating peptide is attached to theC-terminus of the GLP-1 C-terminal peptide. In some embodiments, thefusion protein further comprises a cleavable moiety as known in the artbetween the cell-penetrating peptide and the GLP-1 C-terminal peptidethat cleaves off the cell-penetrating peptide, leaving the GLP-1C-terminal peptide intact.

Peptidomimetics

In some embodiments, the peptides disclosed herein can be modifiedaccording to the methods known in the art for producing peptidomimetics.See, e.g., Kazmierski, W. M., ed., Peptidomimetics Protocols, HumanPress (Totowa N.J. 1998); Goodman et al., eds., Houben-Weyl Methods ofOrganic Chemistry: Synthesis of Peptides and Peptidomimetics, ThieleVerlag (New York 2003); and Mayo et al., J. Biol. Chem., 278:45746(2003). In some cases, these modified peptidomimetic versions of thepeptides and fragments disclosed herein exhibit enhanced stability invivo, relative to the non-peptidomimetic peptides.

Methods for creating a peptidomimetic include substituting one or more,e.g., all, of the amino acids in a peptide sequence with D-amino acidenantiomers. Such sequences are referred to herein as “retro” sequences.In another method, the N-terminal to C-terminal order of the amino acidresidues is reversed, such that the order of amino acid residues fromthe N terminus to the C terminus of the original peptide becomes theorder of amino acid residues from the C-terminus to the N-terminus inthe modified peptidomimetic. Such sequences can be referred to as“inverso” sequences.

Peptidomimetics can be both the retro and inverso versions, i.e., the“retro-inverso” version of a peptide disclosed herein. The newpeptidomimetics can be composed of D-amino acids arranged so that theorder of amino acid residues from the N-terminus to the C-terminus inthe peptidomimetic corresponds to the order of amino acid residues fromthe C-terminus to the N-terminus in the original peptide.

Other methods for making a peptidomimetic include replacing one or moreamino acid residues in a peptide with a chemically distinct butrecognized functional analog of the amino acid, i.e., an artificialamino acid analog. Artificial amino acid analogs include beta-aminoacids, beta-substituted beta-amino acids (“beta3-amino acids”),phosphorous analogs of amino acids, such as a-amino phosphonic acids andb-amino phosphinic acids, and amino acids having non-peptide linkages.Artificial amino acids can be used to create peptidomimetics, such aspeptoid oligomers (e.g., peptoid amide or ester analogues),beta-peptides, cyclic peptides, oligourea or oligocarbamate peptides; orheterocyclic ring molecules. Exemplary retro-inverso peptidomimeticsinclude RGKVL (SEQ ID NO: 8), GRGKVL (SEQ ID NO: 9), or RGRGKVL (SEQ IDNO: 10), wherein the sequences include all D-amino acids.

Modifications

The peptide sequences described herein can be modified, e.g., bymodification of one or more amino acid residues of a peptide by chemicalmeans, either with or without an enzyme, e.g., by alkylation, acylation,ester formation, amide formation, e.g., at the carboxy terminus, orbiotinylation, e.g., of the amino terminus. In some embodiments, thepeptides are modified by the addition of a lipophilic substituent (e.g.,a fatty acid) to an amino acid, e.g., to the Lysine. In someembodiments, the peptides include one or more of an N-terminal imidazolegroup, or a C-terminal amide group. In some embodiments, theepsilon-amino group of Lys34 is substituted with a lipophilicsubstituent, e.g., of about 4-40 carbon atoms, e.g., 8-25 carbon atoms.Examples include branched and unbranched C6-C20 acyl groups. Exemplarylipophilic substituents, and methods of attaching the same (includingvia an optional linker) are provided in U.S. Pat. No. 6,268,343 andKnudsen et al., J. Med. Chem. 43:1664-1669 (2000). In some embodiments,the lipophilic substituent is a fatty acid selected from the groupconsisting of straight-chain or branched fatty acids, e.g., oleic acid,caprylic acid, palmitic acid, and salts thereof.

In some embodiments, the peptide sequences are modified by substitutingone or more amino acid residues of the parent peptide with another aminoacid residue. In some embodiments, the total number of different aminoacids between the sequence-modified peptide and the corresponding nativeform of the GLP-1 C-terminal peptide is up to five, e.g., up to fouramino acid residues, up to three amino acid residues, up to two aminoacid residues, or one amino acid residue.

In some embodiments, the total number of different amino acids does notexceed four. In some embodiments, the number of different amino acids isthree, two, or one. In order to determine the number of different aminoacids, one should compare the amino acid sequence of thesequence-modified GLP-1 peptide derivative with the corresponding nativeGLP-1 C-terminal fragment.

A number of suitable GLP-1 sequence analogues and modifications aredescribed in the art, see, e.g., EP 0708179; WO 91/11457; U.S. Pat. No.6,268,343).

Nucleic Acids, Vectors, and Host Cells

In one aspect, the invention includes nucleic acids encoding a GLP-1 Cterminal peptide or modified peptide as described herein. For example,the invention includes nucleic acids encoding peptides that include asequence set forth herein, e.g., the sequence SEQ ID NO:1 or 2. Nucleicacids disclosed herein also include nucleic acids encoding certainmodified GLP-1 C-terminal pentapeptides, e.g., retro-GLP-1 C-terminalpentapeptides, GLP-1 C-terminal pentapeptides linked to a cellularinternalization (carrier) sequence, and retro-GLP-1 C-terminalpentapeptides linked to a carrier sequence.

Nucleic acids disclosed herein also include both RNA and DNA, includinggenomic DNA and synthetic (e.g., chemically synthesized) DNA. Nucleicacids can be double-stranded or single-stranded. Nucleic acids can besynthesized using oligonucleotide analogs or derivatives (e.g., inosineor phosphorothioate nucleotides). Such oligonucleotides can be used, forexample, to prepare nucleic acids with increased resistance tonucleases.

Also included in the invention are genetic constructs (e.g., vectors andplasmids) that include a nucleic acid encoding a peptide describedherein operably linked to a transcription and/or translation sequencethat enables expression of the peptide, e.g., expression vectors. Aselected nucleic acid, e.g., a DNA molecule encoding a peptide describedherein, is “operably linked” to another nucleic acid molecule, e.g., apromoter, when it is positioned either adjacent to the other molecule orin the same or other location such that the other molecule can directtranscription and/or translation of the selected nucleic acid.

Also included in the invention are various engineered cells, e.g.,transformed host cells, which contain a nucleic acid disclosed herein. Atransformed cell is a cell into which (or into an ancestor of which) hasbeen introduced, by means of recombinant DNA techniques, a nucleic acidencoding a peptide described herein that binds HSP-90 and/or inducesapoptosis in a tumor cell. Both prokaryotic and eukaryotic cells, e.g.,mammalian cells (e.g., tumor cell), yeast, fungi, and bacteria (such asEscherichia coli), can be host cells. An engineered cell exemplary ofthe type included in the invention is a tumor cell that expresses aGLP-1 C-terminal peptide.

Methods of Treatment

The methods described herein include methods for the treatment ofobesity and disorders associated with obesity, e.g., diabetes andmetabolic syndrome; steatotic disease, e.g., hepatic steatosis; andhypertrigylceridemia. In some embodiments, the disorder is diet-inducedobesity, e.g., high-calorie or high-fat diet induced obesity. Generally,the methods include administering a therapeutically effective amount ofa GLP-1 C-terminal peptide or peptidomimetic as described herein, to asubject who is in need of, or who has been determined to be in need of,such treatment.

As used in this context, to “treat” means to ameliorate at least onesymptom of obesity or a disorder associated with obesity. Often, obesityresults in hyperglycemia; thus, a treatment can result in a reduction inblood glucose levels and a return or approach to normoglycemia.Administration of a therapeutically effective amount of a compounddescribed herein for the treatment of obesity will result in decreasedbody weight or fat.

Administration of a therapeutically effective amount of a compounddescribed herein for the treatment of fatty liver disease (FLD) willresult in, e.g., a decrease or stabilization of fat levels in the liver;a decrease or stabilization of inflammation levels in the liver; or areduction, delay or prevention of development of NASH, fibrosis,cirrhosis, or liver failure. In some embodiments, administration of atherapeutically effective amount of a compound described herein for thetreatment of FLD will result in decreased or no increase inintra-cytoplasmic accumulation of triglyceride (neutral fats), and animprovement or no decline in liver function.

Diabetic and Pre-Diabetic Subjects

In some embodiments, the subjects treated by the methods describedherein have diabetes, i.e., are diabetic. A person who is diabetic hasone or more of a Fasting Plasma Glucose Test result of 126 mg/dL ormore; a 2-Hour Plasma Glucose Result in an Oral Glucose Tolerance Testof 200 mg/dL or more; and blood glucose level of 200 mg/dL or above. Insome embodiments, the subjects treated by the methods described hereinare being treated for diabetes, e.g., have been prescribed or are takinginsulin, meglitinides, biguanides, thiazolidinediones, oralpha-glucosidase inhibitors.

In some embodiments the subjects are pre-diabetic, e.g., they haveimpaired glucose tolerance or impaired fasting glucose, e.g., asdetermined by standard clinical methods such as the intravenous glucosetolerance test (IVGTT) or oral glucose tolerance test (OGTT), e.g., avalue of 7.8-11.0 mmol/L two hours after a 75 g glucose drink forimpaired glucose tolerance, or a fasting glucose level (e.g., beforebreakfast) of 6.1-6.9 mmol/L.

The pathogenesis of type 2 diabetes is believed to generally involve twocore defects: insulin resistance and beta-cell failure (Martin et al.,Lancet 340:925-929 (1992); Weyer et al., J. Clin. Invest. 104:787-794(1999); DeFronzo et al., Diabetes Care. 15:318-368 (1992)). Importantadvances towards the understanding of the development of peripheralinsulin resistance have been made in both animal models and humans(Bruning et al., Cell 88:561-572 (1997); Lauro et al., Nat. Genet.20:294-298 (1998); Nandi et al., Physiol. Rev. 84:623-647 (2004);Sreekumar et al., Diabetes 51:1913-1920 (2002); McCarthy and Froguel,Am. J. Physiol. Endocrinol. Metab. 283:E217-E225 (2002); Mauvais-Jarvisand Kahn, Diabetes. Metab. 26:433-448 (2000); Petersen et al., N. Engl.J. Med. 350:664-671 (2004)). Thus, those subjects who have or are atrisk for insulin resistance or impaired glucose tolerance are readilyidentifiable, and the treatment goals are well defined.

In some embodiments, the methods described herein include selectingsubjects who have diabetes or pre-diabetes. In some embodiments, thefollowing table is used to identify and/or select subjects who arediabetic or have pre-diabetes, i.e., impaired glucose tolerance and/orimpaired fasting glucose.

Fasting Blood Glucose From 70 to 99 mg/dL Normal fasting glucose (3.9 to5.5 mmol/L) From 100 to 125 mg/dL Impaired fasting glucose (pre- (5.6 to6.9 mmol/L) diabetes) 126 mg/dL (7.0 mmol/L) and above on Diabetes morethan one testing occasion Oral Glucose Tolerance Test (OGTT) [exceptpregnancy] (2 hours after a 75-gram glucose drink) Less than 140 mg/dL(7.8 mmol/L) Normal glucose tolerance From 140 to 200 mg/dL Impairedglucose tolerance (pre- (7.8 to 11.1 mmol/L) diabetes) Over 200 mg/dL(11.1 mmol/L) on Diabetes more than one testing occasion

Body Mass Index (BMI)

Obesity increases a subject's risk of developing T2D. BMI is determinedby weight relative to height, and equals a person's weight in kilogramsdivided by height in meters squared (BMI=kg/m²). Acceptedinterpretations are given in Table 2.

TABLE 2 Category BMI Underweight ≤18.5 Normal weight 18.5-24.9Overweight   25-29.9 Obese ≥30  

Thus, the methods described herein can include determining a subject'sheight, determining a subject's weight, and calculating BMI from thevalues determined thereby. Alternatively, the methods described hereincan include reviewing a subject's medical history to determine theirBMI.

In some embodiments, the methods described herein include selectingsubjects who have a BMI of 30 or above (i.e., obese subjects).

Metabolic Syndrome

In some embodiments, the methods include determining whether a subjecthas the metabolic syndrome, and selecting the subject if they do havethe metabolic syndrome, then administering an inhibitory nucleic acid asdescribed herein. Determining whether a subject has the metabolicsyndrome can include reviewing their medical history, or ordering orperforming such tests as are necessary to establish a diagnosis.

The metabolic syndrome, initially termed Syndrome X (Reaven, Diabetes.37(12):1595-1607 (1988)), refers to a clustering of obesity,dyslipidemia, hypertension, and insulin resistance. All components ofthe metabolic syndrome are traditional risk factors for vasculardisease. As used herein, the metabolic syndrome is defined by thepresence of at least 3 of the following: abdominal obesity (excessivefat tissue in and around the abdomen, as measured by waistcircumference: e.g., greater than 40 inches for men, and greater than 35inches for women), fasting blood triglycerides (e.g., greater than orequal to 150 mg/dL), low blood HDL (e.g., less than 40 mg/dL for men,and less than 50 mg/dL for women), high blood pressure (e.g., greaterthan or equal to 130/85 mmHg) and/or elevated fasting glucose (e.g.,greater than or equal to 110 mg/dL). In some embodiments, levels ofthese criteria may be higher or lower, depending on the subject; forexample, in subjects of Asian ancestry; see, e.g., Meigs, Curr. Op.Endocrin. Diabetes, 13(2):103-110 (2006). A determination of thepresence of metabolic syndrome can be made, e.g., by reviewing thesubject's medical history, or by reviewing test results.

Based on data from the Third National Health and Nutrition ExaminationSurvey (NHANES III) approximately 24% of the adults in the United Statesqualify as having the metabolic syndrome (Ford et al., JAMA.287(3):356-359 (2002)). Insulin resistance is now felt to be central inthe pathogenesis of these related disorders.

Fatty Liver Disease (FLD)

Nonalcoholic fatty liver disease (NAFLD) and its most severe form,nonalcoholic steatohepatitis (NASH), are associated with high fat diet,high triglyceride levels, obesity, the metabolic syndrome and type IIdiabetes, and pose an increased risk of cardiovascular disease. NAFLD isan accumulation of fat in the liver that is not a result of excessiveconsumption of alcohol. 15% to 25% of cases of NAFLD progress and areassociated with inflammation and liver damage; this condition isreferred to as NASH. NASH is associated with an increased risk ofdeveloping liver cirrhosis and subsequence complications, includinghepatocellular carcinoma. FLD can be caused by excessive alcoholconsumption (alcoholic hepatitis), drugs (such as valproic acid andcorticosteroids (e.g., cortisone or prednisone)), excessive Vitamin A,and obesity. A diagnosis of NAFLD or NASH can be made by methods knownin the art, e.g., by histological examination of liver biopsy samples.

In some embodiments, the methods include determining whether a subjecthas FLD, and selecting the subject if they do have FLD, thenadministering a dose of a GLP-1 C-terminal peptide or peptidomimetic asdescribed herein. Determining whether a subject has FLD can includereviewing their medical history, or ordering or performing such tests asare necessary to establish a diagnosis.

Most individuals with FLD are asymptomatic; the condition is usuallydiscovered incidentally as a result of abnormal liver function tests orhepatomegaly, e.g., noted in an unrelated medical condition. Elevatedliver biochemistry is found in 50% of patients with simple steatosis(see, e.g., Sleisenger, Sleisenger and Fordtran's Gastrointestinal andLiver Disease. Philadelphia: W.B. Saunders Company (2006)). In general,the diagnosis begins with the presence of elevations in liver tests thatare included in routine blood test panels, such as alanineaminotransferase (ALT) or aspartate aminotransferase (AST). Even modest,subclinical increases in hepatic fat accumulation have been shown to bean early component in the progressive pathogenesis of metabolic syndrome(see, e.g., Almeda-Valdes et al., Ann. Hepatol. 8 Suppl 1:S18-24 (2009);Polyzos et al., Curr Mol Med. 9(3):299-314 (2009); Byrne et al., Clin.Sci. (Lond). 116(7):539-64 (2009)).

Imaging studies are often obtained during evaluation process.Ultrasonography reveals a “bright” liver with increased echogenicity.Thus, medical imaging can aid in diagnosis of fatty liver; fatty livershave lower density than spleen on computed tomography (CT) and fatappears bright in T1-weighted magnetic resonance images (Mills). Makinga differential diagnosis of Nonalcoholic Steatohepatitis (NASH), asopposed to simple fatty liver, is done using a liver biopsy. For a liverbiopsy, a needle is inserted through the skin to remove a small piece ofthe liver. NASH is diagnosed when examination of the tissue with amicroscope shows fat along with inflammation and damage to liver cells.If the tissue shows fat without inflammation and damage, simple fattyliver or Nonalcoholic Fatty Liver Disease (NAFLD) is diagnosed. Thus,histological diagnosis by liver biopsy is sought when assessment ofseverity is indicated.

Non-Hepatic Steatosis

Although the liver is most often associated with steatosis, it can occurin any organ, including but not limited to kidneys (renal steatosis,see, e.g., Bobulescu et al., Am J Physiol Renal Physiol. 2008 June;294(6):F1315-22), heart (cardiac steatosis, see, e.g., McGavock et al.,Circulation. 2007 Sep. 4; 116(10):1170-5; McGavock et al., Ann InternMed. 2006 Apr. 4; 144(7):517-24), skeletal muscle, and vasculature(e.g., atherosclerosis); thus, the present methods may also be used totreat those conditions. See, e.g., Federico et al., World JGastroenterol. 2010 Oct. 14; 16(38):4762-72.

Hypertriglyceridemia

Hypertriglyceridemia, or high blood levels of triglycerides, has beenassociated with atherosclerosis, even in the absence ofhypercholesterolemia. Severe hypertriglyceridemia (e.g., levels greaterthan 1000 mg/dL) is also a precursor to pancreatitis. Caused orexacerbated by uncontrolled diabetes mellitus, obesity, and sedentaryhabits, hypertriglyceridemia is a risk factor for coronary arterydisease (CAD). Hypertriglyceridemia is typically diagnosed in thepresence of a fasting plasma triglyceride measurement that is increased,typically above the 90^(th) or 95^(th) percentile for age and sex. TheAdult Treatment Panel III of the National Cholesterol Education Program(JAMA 2001; 285:2486-97) has suggested 4 triglyceride strata in thecontext of assessment of risk of cardiovascular disease: normal (<1.7mmol/L), borderline high (1.7-2.3 mmol/L), high (2.3-5.6 mmol/L) andvery high (>5.6 mmol/L). See, e.g., Yuan et al., CMAJ, 176 (8):1113-1120(2007); Durrington, Lancet 362:717 (2003); Ford et al., Arch Intern Med169:572 (2009).

Dosage

An “effective amount” is an amount sufficient to effect beneficial ordesired results. For example, a therapeutic amount is one that achievesthe desired therapeutic effect. This amount can be the same or differentfrom a prophylactically effective amount, which is an amount necessaryto prevent onset of disease or disease symptoms. An effective amount canbe administered in one or more administrations, applications or dosages.A therapeutically effective amount of a therapeutic compound (i.e., aneffective dosage) depends on the therapeutic compounds selected. Thecompositions can be administered one from one or more times per day toone or more times per week; including once every other day. The skilledartisan will appreciate that certain factors may influence the dosageand timing required to effectively treat a subject, including but notlimited to the severity of the disease or disorder, previous treatments,the general health and/or age of the subject, and other diseasespresent. Moreover, treatment of a subject with a therapeuticallyeffective amount of the therapeutic compounds described herein caninclude a single treatment or a series of treatments.

Dosage, toxicity and therapeutic efficacy of the therapeutic compoundscan be determined by standard pharmaceutical procedures in cell culturesor experimental animals, e.g., for determining the LD50 (the dose lethalto 50% of the population) and the ED50 (the dose therapeuticallyeffective in 50% of the population). The dose ratio between toxic andtherapeutic effects is the therapeutic index and it can be expressed asthe ratio LD50/ED50. Compounds which exhibit high therapeutic indicesare preferred. While compounds that exhibit toxic side effects may beused, care should be taken to design a delivery system that targets suchcompounds to the site of affected tissue in order to minimize potentialdamage to uninfected cells and, thereby, reduce side effects.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

Pharmaceutical Compositions and Methods of Administration

The methods described herein include the manufacture and use ofpharmaceutical compositions, which include GLP-1 C-terminal peptidesdescribed herein as active ingredients. Also included are thepharmaceutical compositions themselves.

Pharmaceutical compositions typically include a pharmaceuticallyacceptable carrier. As used herein the language “pharmaceuticallyacceptable carrier” includes saline, solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like, compatible with pharmaceuticaladministration. Supplementary active compounds can also be incorporatedinto the compositions.

Pharmaceutical compositions are typically formulated to be compatiblewith its intended route of administration. Examples of routes ofadministration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration.

Methods of formulating suitable pharmaceutical compositions are known inthe art, see, e.g., the books in the series Drugs and the PharmaceuticalSciences: a Series of Textbooks and Monographs (Dekker, NY). Forexample, solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use can includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It should be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent that delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle, which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying, which yield a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds can be delivered in theform of an aerosol spray from a pressured container or dispenser thatcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer. Such methods include those described in U.S. Pat. No.6,468,798.

Systemic administration of a therapeutic compound as described hereincan also be by transmucosal or transdermal means. For transmucosal ortransdermal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art, and include, for example, for transmucosaladministration, detergents, bile salts, and fusidic acid derivatives.Transmucosal administration can be accomplished through the use of nasalsprays or suppositories. For transdermal administration, the activecompounds are formulated into ointments, salves, gels, or creams asgenerally known in the art.

The pharmaceutical compositions can also be prepared in the form ofsuppositories (e.g., with conventional suppository bases such as cocoabutter and other glycerides) or retention enemas for rectal delivery.

In some embodiments, the GLP-1 C-terminal peptides are formulated with acell penetrating agent as known in the art, e.g., liposomes or micelles.Biodegradable microparticle or nanoparticle delivery systems thatincrease intracellular uptake, e.g., polymeric and surface modifiednanoparticles as described in US 2009/0136585 and, can also be used.Examples include poly DL-lactide-co-glycolide (PLGA) nanoparticles,e.g., surface-modified with known surface-modifying agents, such asheparin, dodecylmethylammonium bromide (DMAB), DEAE-Dextran, lipofectin,and fibrinogen (see, e.g. Song et al., J. Control. Release, 54:201-211(1998); Labhasetwar et al., J. Pharm. Sci., 87:1229-34 (1998); Lee etal., Biomaterials 29(9):1224-1232 (2008); and US 2009/0136585.

In one embodiment, the therapeutic compounds are prepared with carriersthat will protect the therapeutic compounds against rapid eliminationfrom the body, such as a controlled release formulation, includingimplants and microencapsulated delivery systems. Biodegradable,biocompatible polymers can be used, such as ethylene vinyl acetate,polyanhydrides, polyglycolic acid, collagen, polyorthoesters, andpolylactic acid. Such formulations can be prepared using standardtechniques, or obtained commercially, e.g., from Alza Corporation andNova Pharmaceuticals, Inc. Liposomal suspensions (including liposomestargeted to selected cells with monoclonal antibodies to cellularantigens) can also be used as pharmaceutically acceptable carriers.These can be prepared according to methods known to those skilled in theart, for example, as described in U.S. Pat. No. 4,522,811.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Example 1

GLP-1-Derived Pentapeptide GLP-1(32-36)amide Increases Basal EnergyExpenditure, Inhibits Weight Gain, the Development of InsulinResistance, and Hepatic Steatosis in Diet-Induced Obese Mice

Because the C-terminal pentapeptide, GLP-1(32-36)amide, LVKGRamide, wasalso shown to be a major end-product of the proteolysis of GLP-1 byneprilysin [7] in addition to the nonapeptide GLP-1(28-36)amide,FIAWLVKGRamide, the actions of the pentapeptide, LVKGRamide [8], wereinvestigated in diet-induced obese mice that develop the metabolicsyndrome and in isolated insulin-resistant mouse hepatocytes. Similar tothe nonapeptide reported on earlier [17,18], the pentapeptide curtailsweight gain, inhibits the development of insulin resistance, diabetes,hepatic steatosis, and increases basal energy expenditure in thediet-induced mouse model of metabolic syndrome and suppresses glucoseproduction and ROS levels in isolated insulin-resistant mousehepatocytes. These findings of novel insulin-like actions ofGLP-1-derived C-terminal penta and nona peptides in obese mice andisolated hepatocytes suggest the possibility that they may prove to beuseful treatments for obesity-related diabetes and the metabolicsyndrome.

Materials and Methods

Reagents

GLP-1(32-36)amide, LVKGRamide, was prepared by solid phase peptidesynthesis in the MGH Biopolymers Core Laboratory. The peptide was >98%valid peptide by HPLC and mass spectrometry analyses. Osmotic pumps(Alzet #1004 osmopumps) were from Alzet, Cupertino, Calif. Otherreagents were from Sigma-Aldrich, St Louis, Mo.

Mice

Male C57bl/6 mice at 6 of age were placed on a very high-fat diet (VHFD,60% fat, Research Diets) for 17 weeks. At week 17 the mice on thehigh-fat diet, corresponding to 23 weeks of age, mini-osmopumpscontaining either vehicle or GLP-1(32-36)amide were implantedsubcutaneously for delivery of peptide or vehicle over 16 weeks. Fortynanomoles (20 micrograms) of GLP-1(32-36) was diluted in salinecontaining 0.1% human serum albumin and were infused at a rate of fortynanomoles/kgBW/day for 16 weeks to achieve an estimated concentration ofapproximately 100 pM similar to that reported by infusions ofGLP-1(7-36)amide [20]. For infusions longer than 4 weeks additionalosmopumps with peptide were implanted at the end of each 4 weeksinfusion. Body weights were recorded weekly. Food consumption wasassessed every 3 to 4 days by weight. Energy intake (kcal/gm BW/week)and Feed Efficiency Index (FEI) were evaluated during the infusions ofvehicle or peptide. The latter provides a measure of the efficiency ofcaloric conversion to body weight and it is calculated by determiningthe grams of body weight gain per cage/Kcal of food consumed per cage,[21]. There was no observable change in the activities of the miceamongst the various experimental groups. Mouse care was conducted underapproval by the MGH Institutional Animal Care Use Committee.

Metabolic Parameters Determined in a Closed Metabolic System

Mice were single-caged for three days acclimatization and thentransferred to single-cage metabolic chambers in a PhenoMaster/LabMastersystem (TSE-Systems, Inc., Chesterfield, Mo.) for measurements of oxygenconsumption, CO2, physical activity, and food intake during light anddark cycles for a period of 72 hrs.

Dual Energy X-Ray Absorptiometry (DXA)

Mice were anesthetized with 0.02 ml of a 2% tribromoethanol solution pergram of body weight and scanned with a dual X-ray apparatus (LunarPiximus, GE Medical Systems, Wauwatosa, Wis.). Total, fat, and lean bodymass was quantitatively determined.

Electron Spray and Liquid Chromatography Mass Spectrometry

Mice were given single i.v. injections of the propeptide,GLP-1(9-36)amide and blood samples were taken at times 2, 5, and 10 min.Plasma was assayed for the presence of GLP-1(9-36)amide,GLP-1(28-36)amide, and GLP-1(32-36)amide by electron spray massspectrometry (ES-MS. Pooled plasma samples collected after a 28 daycontinuous infusion of GLP-1(9-36)amide or GLP-1(28-36)amide in micewere assayed for peptide concentrations by liquid chromatography massspectrometry (LC-MS).

Plasma Glucose and Insulin Measurements

Plasma obtained by tail nick from mice fasted for 16 hrs was assayed forglucose by using the one touch ultra-mini glucometer (Life Scan, Johnsonand Johnson company) and for insulin with a rat/mouse Elisa kit (CrystalChem, Downers Grove, Ill.).

Analyses of Liver Samples for Lipid Accumulation and TriglycerideContent.

Representative samples (2-3 gms) of livers were obtained and quickfrozen on solid CO2 at the time of sacrifice and necropsy. The sampleswere subsequently homogenized and triglycerides were extracted andmeasured using a colorimetric enzymatic assay (Serum TriglycerideDetermination kit, Sigma).

Isolated Mouse Hepatocytes

C57bl/6J mice from 10-12 weeks of age were purchased from JacksonLaboratories, Bar Harbor, Me. Diet-induced obesity mice (DIO) wereobtained after C57bl/6J mice of 10-12 weeks of age were fed a high-fatdiet (60% kcal fat, D12492, Research Diets, New Brunswick, N.J.) for 9weeks. Mice were fasted overnight (16 hrs) and primary hepatocytes wereisolated from the livers using a collagen and perfusion gradientpurification [15]. Cells were first seeded using a Dulbecco's modifiedEagle's medium (DMEM) supplemented with 10% FBS, 1 g/L glucose, 2 mmol/1sodium pyruvate, 1 micromol/L dexamethasone, and 0.1 micromol/L insulin,and later maintained in DMEM with 0.2% BSA, 1 g/L glucose, 0.1micromol/L dexamethasone, and 1 nmol/L insulin. Mice were housed andtreated in accord with the regulations of the MGH Institutional AnimalCare Utilization Committee.

Glucose Production Assay

Primary hepatocytes (2×10⁵ cells per well in twelve-well plates) werepre-treated with GLP-1(32-36)amide for 1 hour followed by thestimulation of insulin resistance by the addition of cAMP (10microM)/dexamethasone (50 mM)/sodium lactate (2 mM) in glucose-free DMEMwithout phenol red. The culture media were collected for measuringglucose concentration with a colorimetric glucose assay kit (Sigma). Thereadings were then normalized to total protein content determined fromwhole-cell lysates.

Glucose Tolerance Test (ipGTT) and Fasting Insulin Test Protocol

Mice were maintained in a normal light/dark cycle according to thestandard protocols of the MGH Animal Care and Use Committee. Mice weretested with age matched or litter mate controls and 6 mice per group arerequired. Mice were fasted for 16 hours: beginning around 6 pm theevening prior to the GTT mice are transferred to a new cage with waterbut no food and a DO NOT FEED card is placed in the cardholder. Thefollowing morning the mice were prepared for the glucose tolerance test:animals were weighed, the tail was nicked with a fresh razor blade by ahorizontal cut of the very end, ˜35 to 50 microliters of blood ismassaged from the tail to an eppendorf tube which is immediately placedon ice, baseline blood glucose is measured by the glucose oxidase methodusing a Glucometer Elite glucometer, and 2 grams/kg body weight of 20%D-glucose is drawn up in a Beckton Dickenson D 29 gage ½″ insulinsyringe (one unit of D-glucose for every gram of body weight). Animalswere then transferred to individually labeled 1000 cc cardboard soupcups with the lid liners removed.

Following animal preparation, glucose was injected into theintraperitoneal cavity. At 10, 20, 30, 60, 120, and 140 minutes bloodglucose is sampled from the tail of each mouse by gently massaging asmall drop of blood onto the glucometer strip. Glucose injections andblood glucose sampling is timed to take approximately the same amount oftime per animal (i.e. 25 animals are injected in 12 minutes and bloodglucose sampling of those same 25 animals should also take about 12minutes) so that the sample times are accurate for each animal.

Fasting immunoreactive insulin levels: whole blood samples were spun ina refrigerated microfuge at 14,000 rpm for 10 minutes and transferred toa clean tube. 12 microliters of serum was tested using an ELISA assay(Crystal Chem) with mouse insulin as a standard according to thestandard protocol that comes with the kit.

Reactive Oxygen Species (ROS) Formation Assay (Isolated PrimaryHepatocytes)

Primary hepatocytes from diet-induced obese (DIO) and C57BL/6J mice wereseeded in 12-well plates at a density of 2×105/well for 24 h. C57BL/6Jhepatocytes were treated with 30 mM glucose in the absence or presenceof GLP-1(32-36)amide for 20 to 24 h and compared to DIO hepatocytestreated with GLP-1(32-36)amide. Cells were washed twice in HBSS (Hanks'Balance Salt Solution) and incubated with 10 uM 5-(and6)-carboxy-2′,7′-dichlorohydro-fluorescein diacetate (CM-H2DCFDA) for 45minutes. The media were removed and cells were lysed. ROS was measuredin the cell lysates using a spectrofluorometer (485 nm/535 nm). Datawere normalized to values obtained from untreated controls.

Reactive Oxygen Species (ROS) Formation Assay (H4IIe Hepatoma Cells)

H4IIe cells were seeded in 12-well plates at a density of 1×104/well andincubated overnight in media containing 5.0 mM glucose. Media was thensupplemented with 0.20 mM palmitate in BSA, with and without 100 pMGLP-1(32-36)amide and incubated for an additional 24 h. IntracellularROS was measured by 5-(and 6)-carboxy-2′,7′-dichlorohydro-fluoresceindiacetate (CM-H2DCFDA) (Molecular Probes) as follows. Cells were washedtwice in HBSS (Hanks' Balance Salt Solution) and incubated with 10 uMCM-H2DCFDA for 45 minutes. The media were removed and cells were lysed.ROS was measured in the cell lysates using a spectrofluorometer (485nm/535 nm). Data were normalized to values obtained from the cellstreated with palmitate. BSA n=14. Palmitate n=13. 32-36/Palmitate n=12.

Statistical Analyses

The data are presented as the mean±SE. Statistical analysis wasperformed using Student's t-test. P values of less than 0.05 wereconsidered statistically significant.

Results

GLP-1(32-36)Amide Attenuates Weight Gain in High Fat-Fed Mice.

The continuous infusion of GLP-1(32-36)amide for fourteen weeks (31weeks on the very high far diet) curtailed the rate of weight gain inmice fed VHFD (FIG. 1A). The inhibition of weight gain reachedstatistical significance by week five and it was maintained until theend of the twelve weeks of infusion. The body weight began to decreaseat week 10 of the infusion of the pentapeptide corresponding to theleveling off of weight gain in the vehicle-treated control mice havingreached a near maximum mean body weight of 50-55 gms for the C57bl/5 Jmice on a high-fat diet [22, 23], The average weekly change in bodyweight gain of mice receiving peptide was 50% less than that of the micereceiving control vehicle, measured over ten weeks (FIG. 1B).

Measurements of body lean and fat mass by dual energy X-rayabsorptiometry (DXA) immediately after week sixteen of the controlvehicle and pentapeptide infusions showed a 40% reduction in fat mass inthe peptide infused mice compared to control mice and no significantchanges in lean mass (FIG. 2A). Infusion of the peptide also decreasedtotal body mass (body weight) (FIG. 2B), the relative (%) fat masscompared to vehicle-treated mice (FIG. 2C), and the ratio of fat mass tobody weight (FIG. 2D).

GLP-1(32-36)Amide Increases Energy Intake in VHFD-Fed Mice.

Measurements of food intake (energy intake) in the mice fed VHFD duringten weeks of infusion (weeks 2 to 10) reveals that the mice receivingthe GLP-1(32-36)amide and vehicle infusions consume approximately thesame number of calories: Vehicle, 1.11±0.025 Kcal/g body weight/week;GLP-1(32-36)amide, 1.15±0.030 Kcal/g body weight/week. The FeedEfficiency, an index of the caloric intake distributed into body weight,was decreased by approximately 50% in the peptide-infused obese micecompared to the control obese mice receiving infusion of vehicle alone(FIG. 3). These results suggest that a substantial proportion of theexcessive energy intake was dissipated (burned) rather than going intobody weight by the infusion of the peptide.

Analyses of Metabolic Parameters in a Closed System Shows thatGLP-1(32-36)Amide Increases Basal Energy Expenditure Independently ofPhysical Activity

Obese mice fed the VHFD for 31 weeks and receiving continuous infusionsof GLP-1(32-36)amide or vehicle control for twelve weeks were monitoredfor three days in metabolic chambers. The mice were single caged.Parameters measured were body weight, oxygen consumption VO2,respiratory exchange rate, energy expenditure, home cage activity,drinking and feeding, and urine and feces production. The body weightsheld steady over the three days with vehicle-infused versuspentapeptide-infused mice averaging 52 grams and 38 grams, respectively(FIG. 4A). The whole body oxygen consumption (Total VO2) wassignificantly higher in the pentapeptide-infused mice compared to thecontrol vehicle-infused mice for both the light cycle (P<5.9 E-43) andthe dark cycle (P<4.0 E-66) (FIG. 4B). In addition, mice infused withGLP-1(32-36)amide showed an increase in resting oxygen consumption bothduring the light cycle (P<5.9 E-43) and the dark cycle (P<4.0 E-66)(Figure D and 4E). The average physical activities during both light anddark cycles were not significantly different between the mice receivingpentapeptide versus mice receiving control vehicle (FIG. 4C). Thus, theincrease in VO2, a measure of energy expenditure was not due to changesin physical activity and is attributable to endogenous basal energyexpenditure.

GLP-1(32-36)Amide Attenuates the Development of Fasting Hyperglycemiaand Hyperinsulinemia and Insulin Resistance in High Fat Fed Mice.

Fasting (16 hrs) plasma glucose and insulin levels were determined inthe mice fed the high-fat diet (VHFD) for 15 weeks (21 weeks of age),two weeks before beginning the pentapeptide infusions, and again after12 weeks of continuous infusion of the pentapeptide. at which time themice were on the high-fat diet for 29 weeks (35 weeks of age). After 15weeks on the high-fat diet both the plasma glucose and plasma insulinlevels were elevated in both groups of mice; six mice destined toreceive control vehicle infusions and six mice destined to receiveinfusions of the pentapeptide. Plasma glucose levels were 4 to 5 mM(normal range 3 to 4 mM) and plasma insulin levels were 120 to 200 pM(normal range 40 to 50 pM). By 29 weeks on diet (after 12 weeks ofcontinuous infusion of control vehicle) the fasting plasma glucose andinsulin levels increased to 8.8 mM and 300 pM, respectively. In contrastthe mice receiving the continuous infusion of pentapeptide fastingplasma glucose levels were close to the normal range (5.5 mM) and plasmainsulin levels were somewhat lower (250 pM), (FIGS. 5A and 5B) althoughnot significantly different from the control vehicle level of 300 pM.The effectiveness of the pentapeptide infusion to lower fasting plasmaglucose to the near normal range without an increase in plasma insulinlevels indicates that the pentapeptide improved total body insulinsensitivity. Thus, the infusion of the pentapeptide in insulin-resistantdiet-induced obese mice improves insulin sensitivity similar to thefindings reported on the infusions of the 29 amino acid peptide,GLP-1[9-36)amide [16] and the nonapeptide, GLP-1(28-36)amide [17] indiet-induced obese, insulin-resistant mice. FIGS. 5C and 5D show plasmaglucose (5C) and plasma insulin (5D) after an intraperitoneal glucoseadministration (2 mg/Kg BW ip) in overnight fasted mice fed in VHFD for24-weeks and 16-weeks of continuous infusions of vehicle orGLP-1(32-36)amide, showing that GLP-1(28-36)amide treated mice have anormal glycemic response (5C) and a strong insulin response (5D),whereas the obese vehicle control mice have glucose intolerance and aloss of GSIS (glucose-stimulated Insulin secretion).

GLP-1(32-36)Amide Infusions Result in a Reduction of TriglycerideAccumulation in the Livers of High Fat Fed Mice.

Livers from control mice fed a normal low-fat diet (LFD) and mice fed ahigh-fat diet (VHFD) infused with either GLP-1(32-36)amide or controlvehicle alone were analyzed for contents of triglycerides. The micereceiving infusions of the pentapeptide were decreased by 65%; 35% ofthe vehicle control set at 100% (FIG. 6A) The livers of mice fed VHFDand received the control vehicle infusion had elevated triglyceridelevels (60.5+/−12.1 mg/mg protein). The infusion of GLP-1(32-36)amide tomice fed the high-fat diet (VHFD) diminished the triglycerideaccumulation by 65% to 21.1+/−5.6 mg/mg protein compared to controlvehicle infusion, a reduction to that of the triglyceride content ofnormal mice fed a regular low fat diet (LFD (21.6+/−7) (FIG. 6B). FIG.6C is a table showing continuous infusion of GLP-1(32-36)amide for16-weeks improves plasma circulating levels of glycerol and triglyceridein mice fed on a 60% fat diet, called VHFD, very high-fat diet (ResearchDiets) for 24-weeks. *p<0.02, peptide vs. vehicle; **p<0.008 peptide vs.vehicle. Thus, GLP-1(32-36)amide improves lipid profile.GLP-1(32-36)amide prevents or reverses the first three of themanifestations of the metabolic syndrome, defined as obesity, diabetes,hyperlipidemia, and hypertension.

Mass Spectroscopy of Mouse Plasma Samples Indicate Rapid Conversion ofGLP-1(9-36)Amide to the Nona and Pentapeptides by an Endopeptidase(s) inthe Circulation.

To determine whether the nona and pentapeptides derived-from theC-terminus of GLP-1 are formed in the circulation of mice the propeptideGLP-1(9-36)amide, a product of the cleavage of the parent GLP-1 peptide,GLP-1(7-36)amide, by the diaminopeptidyl peptidase Dpp4, theGLP-1(9-36)amide was pulse-injected intravenously in mice. Plasmasamples taken at 2 min after the injections were analyzed by electronspray mass spectrometry for the presence of the nona and pentapeptides(FIGS. 7B & C). Both peptides were readily detected in the plasma ofmice injected with GLP-1(9-36)amide and not in the plasma of shaminjected mice (FIG. 7C). These findings support the notion thatGLP-1(9-36)amide, the major currently known circulating form of GLP-1,is rapidly converted to the nona and pentapeptides. Since it is knownthat the endopeptidase neprilysin (NEP 24.11) cleaves GLP-1 at sitesbetween amino acids E27 and F28, and between W31 and L32 [7] (FIG. 7A),it seems reasonable to assume that the rapid appearance of the nona andpentapeptides in the circulation of mice by 2 min after the injection ofGLP-1(9-36)amide occurs by the cleavages of GLP-1(9-36)amide. Further itseems reasonable to assume that the cleavages occur via theendopeptidase NEP 24.11, an enzyme known to exist in the circulations ofmice and humans [24].

The plasma concentrations achieved during the continuous infusions ofGLP-1(9-36)amide and GLP-1(28-36)amide were also determined by LC-MS.Plasma samples were pooled from terminal mice after four weeks ofcontinuous infusions of peptides. GLP-1(9-36)amide was not detectable.However, the levels of GLP-1(28-36)amide in the mice infused withGLP-1(9-36)amide were ˜100 pM and the mice infused withGLP-1(28-36)amide, at a concentration of 2.0 mg/ml in the osmopump, hadplasma levels of ˜240 pM. Thus plasma levels of the penta andnonapeptides achieved during the infusions of either GLP-1(9-36) made orGLP-1(28-36)amide were in the range of 100 pM to 240 pM. Since thenormal circulating concentrations of total GLP-1 in the circulation,˜80% of which is GLP-1(9-36)amide, is 20-100 pM, the levels ofnonapeptide achieved in the continuous infusion experiments are onlymodestly super-physiologic.

GLP-1(32-36)Amide Inhibits Glucose Production in Isolated MouseHepatocytes.

To determine whether there may be effects of GLP-1(32-36)amide onmitochondrial functions of oxidative phosphorylation in hepatocytes,gluconeogenesis was examined because uncontrolled hepatic gluconeogensisis an important contributor to fasting hyperglycemia ininsulin-resistant diabetic individuals. Gluconeogenesis was stimulatedin the isolated mouse hepatocytes by the addition of cAMP,dexamethasone, and lactate as described earlier [15]. The combination ofcAMP, dexamethasone, and lactate induces insulin resistance inhepatocytes [25]. The addition of GLP-1(28-36)amide to the hepatocytesdose-dependently suppressed glucose formation (FIG. 9A). Notably,suppression of glucose production occurred only to the component ofglucose production stimulated by cAMP, dexamethasone and lactate, andnot the basal glucose production, suggesting that the effects of GLP-1in the suppression of hepatocyte glucose production is specific forinsulin resistance.

GLP-1(32-36)Amide Suppresses Oxidative Stress and ROS Formation inIsolated Mouse Hepatocytes.

Because the production of reactive oxygen species (ROS) by mitochondriais believed to be a major trigger for the development of insulinresistance [2], hepatic steatosis [1,26,27], and apoptosis via thestimulation of cytochrome C release and the activation of the caspasecascade [29], the intracellular levels of reactive oxygen species (ROS)were measured in hepatocytes in response to GLP-1(32-36)amide (FIGS. 9Aand 9B). Hepatocytes isolated from the livers of normal mice wereincubated in media containing 30 mM glucose to induce insulin resistanceand oxidative stress (elevated levels of ROS). ROS levels were measuredin hepatocytes using the fluorescent indicator CMH2DCFDA. The additionof GLP-1(32-36)amide ((10 pM, 100 pM and 100 nM) to the hepatocytesduring their incubation in 30 mM glucose, which increased ROS levels,prevented the rise in ROS levels (FIG. 9A).

GLP-1(32-36)Amide Reduces ROS Production in Hepatoma Cells Treated withPalmitate.

FIG. 9B is a bar graph showing inhibition of the production of ROS inH4IIe hepatoma cells. H4IIe hepatoma cells were incubated with 0.2 mMpalmitate in the presence or absence of GLP-1(32-36)amide for 24 hr. ROSproduction was measured in the whole cell lysate, *p<0.005 palmitate vs.BSA, #p<0.03 palmitate vs GLP-1(32-36)a/Palmitate. FIG. 9B showsGLP-1(32-36)amide diminishes palmitate-induced oxidative stress.GLP-1(32-36)amide nearly completely normalizes the increase in cellularROS levels induced by the fatty acid palmitate. The pentapeptide combatsthe excessive energy produced by fatty acid oxidation by uncouplingoxidative phosphorylation and converting the energy to heat and therebyprevents the energy from going into reactive oxygen species such assuperoxides, which peroxidate mitochondrial proteins, damage them,elicit the SOS response (pre-apoptosis) and leads to the activation ofthe intrinsic apoptosis system, i.e., programmed cell death.

In summary, in a mouse model of diet-induced obesity and metabolicsyndrome the pentapeptide LVKGRamide, GLP-1 (32-36)amide, curtailsweight gain, increases basal energy expenditure, increases insulinsensitivity, prevents the development of glucose intolerance, diabetes,hyperlipidemia, and hepatic steatosis.

Type 2 diabetes is associated with hyperinsulinemia and insulinresistance leading to elevated hepatic glucose production,hyperglycemia, and hyperlipidemia. Infusions of the C-terminalpentapeptide LVKGRamide, GLP-1 (32-36)amide, derived from glucagon-likepeptide-1 (GLP-1), in high fat diet-induced obese mice for sixteen weekscurtailed the rate of weight gain as early as five weeks. At the end ofthe sixteen week infusion, body weights of mice infused withGLP-1(32-36)amide were decreased by 50% compared to vehicle control thatcorrelated with a 40% decrease in fat mass with no significantdifference in lean mass. Indirect calorimetric studies showed thatalthough mice infused with GLP-1(32-36)amide exhibited lower cumulativefood consumption, the rate of oxygen consumption was significantlyhigher compared to vehicle control throughout the light and dark cycles,findings consistent with an increase in energy expenditure. Thesemetabolic effects were not associated with changes in physical activity.Moreover, the infusion of GLP-1(32-36)amide for sixteen weeks in highfat-fed mice attenuated the development of diabetes since both plasmaglucose and insulin were decreased close to values obtained in mice feda control diet. Intraperitoneal glucose tolerance tests on mice fed thehigh-fat diet infused with GLP-1(32-36)amide were normal compared toimpaired glucose tolerance seen in vehicle control obese mice. Liversobtained from peptide-treated mice showed less steatosis that correlatedwith a 65% decrease in triglyceride accumulation, equivalent totriglyceride levels in control mice fed a low fat diet. Moreover, plasmatriglyceride glycerol levels were lowered by treatment of the mice withGLP-1(32-36)amide. These findings demonstrate biological actions and arole for the C-terminal pentapeptide, GLP-1(32-36)amide, in thetreatment and improvement of obesity-related diabetes, insulinresistance, hypertriglyceridemia, and hepatic steatosis.

REFERENCES

-   [1] Grattagliano I, Palmieri V O, Portincasa P, Moschetta A,    Palasciano G. Oxidative stress-induced risk factors associated with    the metabolic syndrome: a unifying hypothesis. J Nutr Biochem 2008;    19:491-504.-   [2] Haas J T, Biddinger S B. Dissecting the role of insulin    resistance in the metabolic syndrome. Curr Opin Lipidol 2009;    20:206-210.-   [3] Lovshin, J A and Drucker, D J Incretin-based therapies for type    2 diabetes mellitus. Nat. Rev. Endocrinol. 2009; 5: 262-269.-   [4] Kieffer, T J and Habener, J F. The glucagon-like peptides.    Endocr Rev 1999; 20:876-913-   [5] Plamboeck A, Hoist J J, Carr R D, Deacon C F. Neutral    endopeptidase 24.11 and dipeptidyl peptidase IV are both involved in    regulating the metabolic stability of glucagon-like peptide-1 in    vivo. Adv Exp Med Biol. 2003; 524:303-12.-   [6] Deacon C F. Circulation and degradation of GIP and GLP-1. Horm.    Metab. Res. 2004; 36:761-765.-   [7] Hupe-Sodmann K, McGregor G P, Bridenbaugh R, GOke R, GOke B,    Thole H, Zimmermann B, Voigt K. Characterisation of the processing    by human neutral endopeptidase 24.11 of GLP-1(7-36) amide and    comparison of the substrate specificity of the enzyme for other    glucagon-like peptides. Regul Pept 1995; 58:149-156.-   [8] Tomas E, Habener J F. Insulin-like actions of glucagon-like    peptide-1: A dual receptor hypothesis. Trends Endocrinol Metab 2010;    2159-2167.-   [9] Nikolaidis, L A, Elahi D, Shen, Y T, Shannon, R P. Active    metabolite of GLP-1 mediates myocardial glucose uptake and improves    left ventricular performance in conscious dogs with dilated    cardiomyopathy. Am. J. Physiol. Heart Circ. Physiol. 2005;    289:H2401-H2408.-   [10] Ban, K, Noyan-Ashraf, M H, Hoefer, J, Bolz, S S, Drucker, D J,    Husain, M. Cardioprotective and vasodilatory actions of    glucagon-like peptide 1 receptor are mediated through both    glucagon-like peptide 1 receptor-dependent and receptor-independent    pathways. Circulation 2008; 117:2340-2350.-   [11] Sonne, D. P., Engstrom, T., Treiman, M. Protective effects of    GLP-1 analogues exendin-4 and GLP-1(9-36) amide against    ischemia-reperfusion injury in rat heart. Regul. Pept. 2008; 146:    243-249.-   [12] Ban K, Kim K H, Cho C K, Sauvé M, Diamandis E P, Backx P H,    Drucker D J, Husain M. Glucagon-like peptide    (GLP)-1(9-36)amide-mediated cytoprotection is blocked by    exendin(9-39) yet does not require the known GLP-1 receptor.    Endocrinology. 2010; 151:1520-1531.-   [13] Green, B D, Hand, K V, Dougan, J E, McDonnell, B M, Cassidy, R    S, Grieve, D J. GLP-1 and related peptides cause    concentration-dependent relaxation of rat aorta through a pathway    involving KATP and cAMP. Arch. Biochem. Biophys, 2008; 478:136-142.-   [14] Elahi, D, Egan, J M, Shannon, R P, Meneilly, G S, Khatri, A,    Habener, J F, Andersen, D K. Glucagon-like peptide-1 (9-36) amide,    cleavage product of glucagon-like peptide-1 (7-36) is a    glucoregulatory peptide. Obesity 2008; 16:1501-1509.-   [15] Tomas E, Stanojevic V, Habener J F. GLP-1(9-36)amide metabolite    and suppression of glucose production in isolated mouse hepatocytes.    Horm Metab Res 2010; 42:657-662.-   [16] Tomas E, Stanojevic V, Wood J A, Habener J F. GLP-1(9-36)amide    metabolite inhibits weight gain and attenuates diabetes and hepatic    steatosis in diet-induced obese mice. Diabetes Obes Metab. 2011;    13:26-33.-   [17] Tomas E, Wood J A, Stanojevic V, Habener J F. GLP-1-derived    nonapeptide GLP-1(28-36)amide inhibits weight gain and attenuates    diabetes and hepatic steatosis in diet-induced obese mice. Regul    Pept. 2011; 169:43-48-   [18] Tomas E, Stanojevic V, Habener, J F. GLP-1-Derived Nonapeptide    GLP-1(28-36)amide targets to mitochondria and suppresses glucose    production and oxidative stress in isolated mouse hepatocytes. Regul    Pept 2011; 167:177-184.-   [19] Abu-Hamdah, R, Rabiee, A, Meneilly, G S, Shannon, R P,    Andersen, D K, Elahi D. Clinical review: The extrapancreatic effects    of glucagon-like peptide-1 and related peptides. J. Clin. Endocrinol    Metab. 2009; 94:1843-1852.-   [20] Zhang J, Tokui Y, Yamagata K, Kozawa J, Sayama K, Iwahashi H,    Okita K, Miuchi M, Konya H, Hamaguchi T, Namba M, Shimomura I,    Miyagawa J I. Continuous stimulation of human glucagon-like    peptide-1 (7-36) amide in a mouse model (NOD) delays onset of    autoimmune type 1 diabetes. Diabetologia. 2007; 50:1900-1999.-   [21] Parekh P I, Petro A E, Tiller J M, Feinglos M N, Surwit R S.    Reversal of diet-induced obesity and diabetes in C57BL/6J mice.    Metabolism. 1998; 47:1089-1096.-   [22] Bartels E D, Bang C A, Nielsen L B. Early atherosclerosis and    vascular inflammation in mice with diet-induced type 2 diabetes. Eur    J Clin Invest. 2009 March; 39(3):190-9.-   [23] Guo J, Jou W, Gavrilova O, Hall K D Persistent diet-induced    obesity in male C57BL/6 mice resulting from temporary obesigenic    diets. PLoS One. 2009; 4(4):e5370.-   [20] Standeven K F, Hess K, Carter A M, Rice G I, Cordell P A,    Balmforth A J, Lu B, Scott D J, Turner A J, Hooper N M, Grant P J.    Neprilysin, obesity and the metabolic syndrome. Int J Obes (Lond).    2010 Nov. 2.-   [25] Liu H Y, Collins Q F, Xiong Y, Moukdar F, Lupo E G Jr, Liu Z,    Cao W. Prolonged treatment of primary hepatocytes with oleate    induces insulin resistance through p38 mitogen-activated protein    kinase. J Biol Chem. 2007 May 11; 282:14205-14212.-   [26] Watanabe S, Yaginuma R, Ikejima K, Miyazaki A. Liver diseases    and metabolic syndrome. J Gastroenterol. 2008; 43:509-518.-   [27] Stein L L, Dong M H, Loomba R. Insulin sensitizers in    nonalcoholic fatty liver disease and steatohepatitis: Current    status. Insulin sensitizers in nonalcoholic fatty liver disease and    steatohepatitis: Current status. Adv Ther 2009; 26:893-907.-   [28] Reddy J K, Rao M S. Lipid metabolism and liver    inflammation. II. Fatty liver disease and fatty acid oxidation. Am J    Physiol Gastrointest Liver Physiol. 2006; 290:G852-8.-   [29] Zhao K, Zhao G M, Wu D, Soong Y, Birk A V, Schiller P W, Szeto    H H. Cell-permeable peptide antioxidants targeted to inner    mitochondrial membrane inhibit mitochondrial swelling, oxidative    cell death, and reperfusion injury. J Biol Chem. 2004; 13;    279:34682-34690.-   [30] Plamboeck, A., Holst, J. J., Carr, R. D., Deacon, C. F. Neutral    endopeptidase 24.11 and dipeptidyl peptidase IV are both mediators    of the degradation of glucagon-like peptide 1 in the anaesthetized    pig. Diabetologia 2005; 48:1882-1890.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method of treating obesity, metabolic syndrome,hepatic steatosis, non-hepatic steatosis, hypertriglyceridemia, ordiabetes in a subject, the method comprising administering atherapeutically effective amount of a composition comprising an isolatedpeptide consisting of a sequence (SEQ ID NO: 3)Leu-Val-(Lys/Arg)-Gly-Arg-Xaa,

wherein Xaa can be Gly, Gly-Arg, Gly-Arg-Gly, or absent, and aphysiologically acceptable carrier, to a subject in need thereof.
 2. Themethod of claim 1, wherein the subject is obese.
 3. The method of claim1, wherein the subject is obese due to consumption of a high fat diet.4. The method of claim 1, wherein the C terminus of the isolated peptideis an Arg and the peptide is amidated.
 5. The method of claim 1, whereinone or more amino acids of the isolated peptide are modified byattachment of a fatty acid.
 6. The method of claim 5, wherein the fattyacid is selected from the group consisting of palmitate and oleate. 7.The method of claim 1, wherein Xaa is absent.
 8. The method of claim 1,wherein Xaa is Gly.
 9. The method of claim 1, wherein Xaa is Gly-Arg.10. The method of claim 1, wherein Xaa is Gly-Arg-Gly.
 11. The method ofclaim 1, wherein the isolated peptide is fused to a cell-penetratingpeptide.
 12. The method of claim 11, wherein the cell-penetratingpeptide is fused on the C-terminus of the peptide of SEQ ID NO:3. 13.The method of claim 11, wherein the cell-penetrating peptide is selectedfrom the group consisting of HIV-derived TAT peptide, penetratins,transportans, SS peptides, and hCT derived cell-penetrating peptides.